Project summary Liquid chromatography-mass spectrometry (LC-MS) has become an influential technology in biomedical investigations. However, absolute quantification of compounds remains a major challenge using this technique. In molecular LC-MS, an analytical standard for each compound is needed for absolute concentration measurements. The standards are often unavailable for many identified compounds, requiring major efforts for their synthesis and purification for analytical purposes. The problem of standard unavailability is frequently encountered in quantitative characterization of xenobiotic metabolism, e.g. in drug development. This proposal addresses quantification without compound-specific standards by advancing elemental MS of F, Cl, P, and S, prevalent heteroatoms in both small and large biologically important molecules. Elemental MS provides compound-independent quantification of elements in LC- separated compounds. The elemental concentrations are then readily translated to molecular concentrations using molecular formulae (confirmed by molecular LC-MS). In the proposed research, analytical performance of elemental MS for heteroatoms is significantly enhanced compared to the existing elemental MS technologies. The improvements arise from developing an elemental ionization method that offers: 1) a new mechanism for efficient ionization of F and Cl, and 2) compatibility with advanced ion separation methods in molecular LC-MS platforms, enabling reduction of isobaric interferences via ultra-high-resolution MS and ion mobility separations previously unavailable in elemental MS. Notably, the second advantage also facilitates adoption of elemental quantification in biomedical investigations by eliminating the need for a dedicated elemental MS platform. Our investigations will be in collaboration with instrument manufacturing and pharmaceutical collaborators from industry, highlighting the impact and adoptability of the proposed technique.